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Corn naringenin methyltransferase gene ZmNOMT and application thereof in plant broad-spectrum disease resistance

A technology of prime methyl and transferase, applied in the direction of transferase, application, plant products, etc.

Pending Publication Date: 2022-07-15
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few studies on the biological function of NOMT. Only OsNOMT was cloned in rice and found that it can catalyze the production of cherry blossom lignin with naringenin (SHIMIZU T, LIN F, HASEGAWA M, et al. Purification and identification of naringenin 7-O-Methyltransferase, akey enzyme in biosynthesis of flavonoid phytoalexin sakuranetin in rice. The Journal of Biological Chemistry, 2012, 287:19315-19325), and the function of NOMT directly involved in plant disease resistance has not yet been reported

Method used

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  • Corn naringenin methyltransferase gene ZmNOMT and application thereof in plant broad-spectrum disease resistance
  • Corn naringenin methyltransferase gene ZmNOMT and application thereof in plant broad-spectrum disease resistance
  • Corn naringenin methyltransferase gene ZmNOMT and application thereof in plant broad-spectrum disease resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. Cloning of ZmNOMT

[0050] 1.1 Extraction of total RNA from maize inbred line B73

[0051] (1) Put the corn B73 leaf material into a high-temperature sterilized mortar, pour it into liquid nitrogen and grind it into powder; continuously add liquid nitrogen during the grinding process to prevent melting;

[0052] (2) Transfer the ground leaf powder into a 1.5ml centrifuge tube, quickly add 1ml RNAiso Plus extract, vortex, and let stand for 10min at room temperature;

[0053] (3) Add 200 μl of chloroform, gently shake and mix until the liquid is stratified, and leave it at room temperature for 5 minutes;

[0054] (4) 4°C, 12,000rpm, centrifuge for 15min, transfer 500μl of supernatant to a new 1.5ml centrifuge tube, add 500μl of isopropanol, invert up and down to mix, and place at room temperature for 10mim;

[0055] (5) 4°C, 12,000rpm, centrifuge for 15min, discard the supernatant, wash the precipitate with 1ml 75% ethanol, flick the bottom of the tube to mak...

Embodiment 2

[0087] Example 2. Construction of ZmNOMT overexpression vector and verification of disease resistance function of transgenic lines

[0088] 2.1 Construction of maize ZmNOMT overexpression vector

[0089] (1) Double enzyme digestion of maize overexpression vector pCAMBIA3301 with BamH1 and Xma1;

[0090] (2) BamH1 and Xma1 restriction sites were added at both ends of the gene ZmNOMT;

[0091] (3) The target band is recovered by gel after double enzyme digestion of the gene, and connected to the pCAMBIA3301 carrier through T4 DNA ligase;

[0092] (4) Transform E. coli competent DH5α, identify the positive clones by colony PCR, and sequence the plasmid in advance. The plasmid vector after correct sequencing is named pCAMBIA3301::ZmNOMT, and its map is shown in Figure 4 .

[0093] 2.2 Plasmid transformation Agrobacterium

[0094] (1) Add 1 μg of plasmid vector to 50 μl of Agrobacterium competent EHA105, mix well, and take an ice bath for 30 minutes;

[0095] (2) Quick-freeze...

Embodiment 3

[0151] Example 3. Construction of ZmNOMT Gene Editing Vector and Verification of Disease Resistance of Edited Lines

[0152] 3.1 Construction of maize ZmNOMT gene editing vector

[0153] The CRISPR / CAS9 base vector was single digested with HindIII. Based on the principle of CRISPR / CAS9 technology and ZmNOMT genome sequence, two target sites, gRNA1 and gRNA2, were designed. The nucleotide sequence of gRNA1 is shown in SEQ ID No.3, and the nucleotide sequence of gRNA2 is shown in SEQ ID No.4. Show. The U-S (U6-gRNA-sgRNA cassette) fragment was used as the template to design specific amplification primers with vector nick homology arms and overlapping PCR overlapping parts (20bp gRNA part), and U-S was used as the template to amplify the target fragment U6-2 promoter And sgRNA scanfold fragment, finally obtained CPB::U6-2::gRNA::sgRNA::CPB overlapping fragment by overlapping PCR, after the overlapping PCR product was sequenced correctly, T4 DNA ligase (TaKaRa) was used to conne...

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PUM

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Abstract

The invention discloses a corn naringenin methyltransferase gene ZmNOMT. The cDNA (complementary deoxyribonucleic acid) nucleotide sequence of the corn naringenin methyltransferase gene ZmNOMT is shown as SEQ ID No.1; the invention also discloses an application of improving the broad-spectrum disease resistance of plants and an application of improving the naringenin content by reducing the expression of the corn naringenin methyltransferase gene ZmNOMT. Experiments prove that through inoculation identification of pathogenic fungi of a ZmNOMT transgenic overexpression positive strain, it is found that the ZmNOMT overexpression positive strain shows a susceptible phenotype to various corn diseases, and it is proved that ZmNOMT is a new susceptible gene. Two target spots are designed for the ZmNOMT by further utilizing a CRISPR / CAS9 technology, gene editing is carried out on the ZmNOMT, and it is found that the naringenin content in a ZmNOMT editing strain is remarkably improved; the ZmNOMT edited strain is subjected to pathogenic bacterium inoculation identification, and the ZmNOMT edited strain shows broad-spectrum disease resistance to various corn fungal diseases. The invention provides a new gene, a new material and a new method for disease-resistant breeding of corn, and the application prospect is wide.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and particularly relates to a corn naringenin methyltransferase gene ZmNOMT and its application to improve broad-spectrum disease resistance of plants by reducing the expression of ZmNOMT. Background technique [0002] Corn (Zea mays L.) is the largest grain crop in my country, as well as an important feed and industrial raw material, occupying an important position in national food security. It is predicted that my country's demand for corn will continue to increase, and the annual growth will exceed 2 million tons. Affected by global climate change, maize diseases are becoming more and more serious, bringing huge losses to maize production. For example, the perennial stem rot in the Huanghuaihai summer corn area, the southern rust epidemic in 1998 and 2021, and the perennial ear rot, large spot and small spot, etc. Therefore, cultivating new varieties of high-quality ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/84A01H5/00A01H6/46
CPCC12N9/1007C12N15/8218C12N15/8282C12N15/8243C12N15/8205C12Y201/01232
Inventor 王官锋李雅洁刘孟洁
Owner SHANDONG UNIV
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